19BT04
DEVELOPMENT OF BIODEGRADABLE CHITOSAN FILM FROM BIO WASTE AND ITS MULTIPLE APPLICATIONS Mr. DHANUSH S KUMAR, Ms. HANNATH RASEENA, Mr. KUNAL SAGAR, Ms.KUSUMA LK (B.E. Biotechnology) Guide: Dr. ANIL KUMAR H.S, Associate Professor Department of Biotechnology Engineering, NMAM Institute of Technology, Nitte, Karkala
INTRODUCTION
ABSTRACT In the present world there is a high demand to develop biodegradable, functional, antimicrobial material. Chitin and chitosan is a cheap most abundant natural polymer owing incomparable properties such as non toxicity, antioxidant, film forming ability. Chitosan is obtained by de-acetylation of chitin.Catalase immobilized chitosan film was able to degrade hydrogen peroxide which was used as a preservative in sea foods. Chitosan based rapid indicator was formulated by coating chemical reagent which is simple detection technique which can detect the presence of hydrogen peroxide and formalin. chitosan film was used as dialysis bag for the separation of proteins in a solution. Chitosan
film
incorporated
with
silver
nanoparticles
and
polyaniline showed electrical conductivity with a simple circuit. Chitosan film which was coated with herbal extract showed wound healing and antimicrobial property and the moisture sensor present
the wound healing patch helps to detect moisture in the wound.
MATERIALS & METHODS Demonstration of Chitosan application Removal of hydrogen peroxide from sea foods using catalase immobilized chitosan film. Preparation of chitosan based rapid indicator strips for detecting formalin and hydrogen peroxide. Preparation of biodegradable selective permeable chitosan membrane for protein dialysis. Preparation of conducting chitosan film Preparation of herbal wound healing smart patch
UV analysis of different concentration of hydrogen peroxide
Fig.14: Working model of CBRI strips for detection of (a) Formalin (b) H2O2 0.20%
0.50% Standard
5% Blank
15%
Sample
Fig.:13 UV analysis of different concentration of hydrogen peroxide
Fig.16: Protein sample dialyzed using 7% chitosan film (a) Standard protein sample (b) Sample inside the dialysis bag (c) Sample outside the dialysis bag
Fig.17: Protein sample dialyzed using 5% chitosan film (a) Standard protein sample (b) Sample inside the dialysis bag (c) Sample outside the dialysis bag
Fig.15: Protein sample dialyzed by 1% chitosan and 1% nanochitosan respectively (a) Sample outside the 1% chitosan dialysis bag (b) Sample inside 1% chitosan dialysis bag (c) Sample outside 1% nanochitosan dialysis bag (d) Sample inside 1% nanochitosan dialysis bag (e) Standard protein sample
Fig.18: Fish treated with (a) no formalin (b) formalin
Fig.20: Moisture sensor embedded herbal wound healing smart patch (a) Top side (b) Drug coated side
METHODOLOGY ◙ Removal of hydrogen peroxide from sea foods using catalase immobilized chitosan film Extraction of catalase from banana peels Immobilization of catalase on chitosan film Determination of immobilized protein and coating of hydrogen peroxide on the fish. CBRI strips to detect formalin and hydrogen peroxide in food samples. Preparation of chitosan film Coating the chemical reagent on the chitosan film Preparation of CBRI strips. Biodegradable selective permeable chitosan membrane for protein dialysis Different concentration of chitosan and nano chitosan were prepared Protein samples with different molecular weight was separated and the efficiency was analyzed ug SDS PAGE electrophoresis. Conducting chitosan film Silver nanoparticle synthesis by chemical method (method 1) Polyaniline preparation and coating on chitosan film (method 2)
MATERIALS & METHODS
◙ Preparation of chitosan film from chitin shrimp shells by deacetylation.
◙ Preparation of catalase immobilized chitosan film to remove hydrogen peroxide from sea foods. ◙ Preparation of chitosan based rapid indicator strips
◙ Deacetylation of chitin – by 60% NaOH ◙ Solubility of chitosan and film production ◙ Antimicrobial property of chitosan film ◙ Preparation of nano chitosan – by adding TPP
for the detection of formalin and hydrogen peroxide from food samples. ◙ Preparation of semi permeable membrane (dialysis) using chitosan and nanochitosan. ◙ Preparation
of
conducting
film
Fig.1: Shrimp shell chitin
using
Fig.3: Chitosan powder
Fig.2: Deacetylation of chitin to chitosan
silver
nanoparticles and polyaniline. ◙ Preparation of wound healing patch and embedding of moisture sensor. Fig.4: Chitosan solution Fig.5: Nanochitosan powder
Fig.6.: Catalase immobilized chitosan film
Fig.7: (a) Adulterated fish wrapped with catalase immobilized chitosan film (b) Adulterated fish without wrapping with any film (c) Adulterated fish wrapped with chitosan film
METHODOLOGY Herbal wound healing smart patch Preparation of herbal extract coating on the chitosan film Antibacterial test for herbal leaf extract and embedding of moisture sensor on the wound healing patch.
RESULTS
Fig.10: Chitosan film Fig.11: Anti-bacterial test for chitosan film using (a) E.coli (b) Bacillus subtilis
Fig.12: Catalase immobilized chitosan film
Table 1: Bradford’s method for protein estimation Total
amount
of Amount of catalase Amount of catalase not
catalase (%) Fig.9: strip indicating (a) Milk sample without adulterant (b) Milk sample with 0.3% hydrogen peroxide (c) Milk sample with 0.01% hydrogen peroxide
Fig.8: Chitosan based dialysis bag
Sample 100%
immobilized (%)
immobilized (%)
65.2%
34.8%
Table 2: fish washings showing hydrogen peroxide concentration in percentage
Hydrogen peroxide concentration in percentage (%) Adulterated
fish
wrapped
with
100%
catalase immobilized chitosan film Fig.9: Moisture sensor embedded smart wound healing patch
Adulterated fish without wrapping
100%
with any film Adulterated
fish
wrapped
with
75%
chitosan film
CONCLUSION
RESULTS 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0
◙ Biowastes are the waste material which readily degradable in both aerobic and anaerobic condition. Biowastes can not only be used as manure for agriculture but can be converted into value added product using using appropriate technology. ◙ Chitosan is a linear polysaccharide produced commercially by de-cetylation of chitin which is one of the component of crustacean exoskeleton ( such as crabs and shrimps). ◙ Now a days there is a huge demand for biodegradeable food packing and preservative detection kit, chitosan due to its film forming and anti-microbial property it can be used for these various purposes. ◙ Chitosan films when coated with enzymes such as catalase, it can help in the removal of preservative like Hydrogen Peroxide. ◙ Chitosan films when coated with chemicals were used to prepare adulterant detection kit. ◙ Chitosan films were also prepared using different concentration which were used to prepare dialysis bag ◙ Due its anti-microbial property, chitosan films can be used in wound healing patch. The prepared wound healing patch was coated with herbal extract having healing ability and embedded with moisture sensor to know the status of the wound.
OBJECTIVES
Fig.19: Simple circuit using polyaniline coated chitosan film
Fig.21: Simple circuit using silver nanoparticle coated chitosan film
DISCUSSION
DISCUSSION
Chitosan films produced can be used for food packaging and the film when immobilized with chitosan has the ability to remove hydrogen peroxide which is used as preservative in fish. Catalase was derived from banana peels. Bardfords estimation was performed to analyse the amount of catalase immobilized on the chitosan. From table 2 it was observed that there was a reduction in hydrogen peroxide concentration in the sample when compared to positive and negative control. From the fig it was observed that even at low concentration of hydrogen peroxide, the catalase immobilised chitosan film could effectively remove the hydrogen peroxide from the adulterated sea foods. Chitosan based rapid indicator strips were prepared using chemically coated chitosan films. These strips can detect adulterants in milk and other food products such as hydrogen peroxide and formalin respectively. These strips are user friendly, no reagents required and very cheap. Cost of each strip is 1 rupee. Dialysis bags were developed using chitosan films. These films were able to dialyse protein sample. Protein confirmation of the dialyzed sample was done using biuret test. Different films were prepared using different chitosan concentration(1%,5%, 7%) and also nano chitosan films were prepared. The dialysed samples of each film were collected and SDS PAGE was carried out from this it was observed that 7% chitosan film and nano chitosan film showed better separation of protein samples. The chitosan films were coated with silver nanoparticle and polyaniline solution respectively to make the chitosan film conductive. The conductivity of the films where confirmed by a simple circuit design were the silver nanoparticle and polyaniline solution coated chitosan films were used as connective wires. A smart wound healing patch was designed using chitosan films coated with herbal extect. From fig. and fig. it was observed that Zanthoxylumrestacould inhibit the growth of bacterial more compared tonaringicrenulata.
Hence Zanthoxylumrestawas used as wound healing material in the herbal wound healing smartThe herbal wound healing smart patch was embedded with moisture sensor along with a switch and a LED. Since the electrode which is a copper wire comes in direct contact with the wound it was replaced by polyaniline coated chitosan film which is conducting and biocompatible in nature. Waterproof adhesive tape was placed on the top of elastic adhesive bandage to make the entire patch waterproof.
• This study exposes the approaches to convert biowaste to useful commercial product. • Deacetylation of chitin was done to obtain chitosan and the chitosan was dissolved in organic solvent for the development of films. • Catalase immobilized chitosan film has dual purpose i.e., it can be used as food packaging and also to remove hydrogen peroxide which is a preservative for seafoods. • Chemically coated chitosan film was used develop CBRI strips which can detect adulterants like hydrogen peroxide and formalin. • Chitosan based dialysis bag was developed which is used for the separation of proteins. • Conducting chitosan film was developed by coating conducing materials like silver nanoparticle and polyaniline. • Moisture sensor embedded herbal wound healing smart patch were developed by coating chitosan film with medicinal herbal extract and conducting chitosan films were used to replace copper electrode in the patch.
REFERENCES • • • • •
.Çetinus, Şenay Akkuş, and H. Nursevin Öztop (2000) "Immobilization of catalase on chitosan film." Enzyme and microbial technology 26.7: 497-501. Daisy Meager (2016) Blog- Italy is bleaching old seafood to make look fresh M. Adlim, Hasan, Zarlaida Fitri, Yulida Amri, Martina Sari and Saiful Mahaya (2011) “Preparation of chemical sensors for simple formalin detection in contaminated food”. Layla O. Farhan, Seenaa A. Mustafa, Areej Sh. Hameed (2016) “Partial Purification and Characterization of Catalase from Banana Peels” Volume: 13 Issue: 2 Pages: 392-398. Abraham, Agil, P. A. Soloman, and V. O. Rejini (2016) "Preparation of chitosanpolyvinyl alcohol blends and studies on thermal and mechanical properties." Procedia Technology 24: 741-748